Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes

Certain cytokines including IFN-gamma possess macrophage-activating factor activity that enhances the ability of these effector cells to destroy intracellular pathogens. A panel of recombinant and highly purified human cytokines was screened to detect this effect on the activation of human monocytes to kill Mycobacterium avium in an in vitro model. Peripheral blood monocytes obtained from 15 healthy donors were precultured for 2 days before infection. Monocytes were infected with two strains of M. avium, one AIDS-associated and relatively avirulent strain (86m2096), and the other a non-AIDS-associated isolate that demonstrated consistent and rapid growth in cultured human monocytes (LR114F). The effects of recombinant and purified human cytokines on M. avium infection were assayed by determining CFU of M. avium in lysates of infected monocytes after 0, 4, and 7 days of culture. After infection, monocytes were cultured in medium alone or continuously in the presence of the following cytokines: IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, granulocyte-macrophage-CSF, or macrophage-CSF. In some experiments, cultures were performed in the presence of indomethacin (IM) in addition to cytokines. Culture in the presence of rIFN-gamma was associated with a decrease in mycobacterial growth within human monocytes. The combination of 300 U/ml of IFN-gamma plus 1 micrograms/ml of IM was associated with a 10-fold decrease (p less than 0.01) in intracellular growth of the virulent strain (LR114F) compared with unstimulated cultures. No other cytokine or combination of a cytokine with IM inhibited the intracellular growth of either strain of M. avium in human monocytes. Rather, several cytokines enhanced the intracellular growth of M. avium. IL-3, IL-6, and macrophage-CSF increased the growth of one, and IL-1 alpha of both strains of M. avium tested. IL-1 alpha and IL-6 also induced M. avium growth in tissue culture medium without monocytes. These studies indicate bidirectional effects of cytokines on intracellular parasitism that may influence the outcome of M. avium infection.     

Recombinant interleukin-6 increases the intracellular and extracellular growth of Mycobacterium avium

Human monocytes were isolated from the peripheral blood of normal donors and allowed to differentiate in vitro into macrophages. The susceptibility of these cells to infection with a virulent Mycobacterium avium and its modulation by some soluble factors was monitored. The virulent strain of Mycobacterium avium grew progressively in untreated macrophage monolayers. Interleukin-6 (IL-6) was tested for its ability to modulate the macrophage-mycobacteria interaction. Surprisingly, IL-6 was shown to increase M. avium growth in macrophage monolayers by twofold as compared with untreated cells, when added before or after infection. Moreover, addition of rIL-6 to replicating mycobacteria in vitro enhanced their growth two- to three-fold as compared with cultures treated with rIL-6 and a rabbit antiserum to rIL-6. Treatment with IL-6 and interferon-gamma (IFN-gamma) or IL-4 did not modify the growth promoting effect of IL-6 in human macrophages. Overall, our results suggest that IL-6 may contribute significantly to the pathogenesis of infections with M. avium by promoting mycobacterial growth.     

Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria

The pathogenesis of tuberculosis (TBC) meningitis is still unknown. As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection. Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis. Results showed that microglia was productively infected by mycobacteria which could grow inside the cells. Mycobacteria internalization was more rapid for M. avium, but M. tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria. TNF-alpha expression was not affected by M. avium, whereas an increase followed by a decrease was observed in M. tuberculosis. Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect. Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response. This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.     

Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and L?wenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.     

Maximum growth rate of Mycobacterium avium in continuous culture or chronically infected BALB/c mice

Mycobacterium avium is a human pathogen which may cause either chronic or disseminated disease and the organism exhibits a slow rate of growth. This study provides information on the growth rate of the organism in chronically infected mice and its maximal growth rate in vitro. M. avium was grown in continuous culture, limited for nitrogen with 0.5 mM ammonium chloride and dilution rates that ranged from 0.054 to 0.153 h-1. The steady-state concentration of ammonia nitrogen and M. avium cells for each dilution rate were determined. The bacterial saturation constant for growth-limiting ammonia was 0.29 mM (4 micrograms nitrogen/ml) and, from this, the maximal growth rate for M. avium was estimated to be 0.206 h-1 or a doubling time of 3.4 h. BALB/c mice were infected intravenously with 3 x 10(6) colony-forming units and a chronic infection resulted, typical of virulent M. avium strains. During a period of 3 months, the number of mycobacteria remained constant in the lungs, but increased 30-fold and 8,900-fold, respectively, in the spleen and mesenteric lymph nodes. The latter increase appeared to be due to proliferation in situ. The generation time of M. avium in the mesenteric lymph nodes was estimated to be 7 days.     

Mycobactins and exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and other related species

Following iron-deficient growth, mycobactins and exochelins were isolated from 11 strains of Mycobacterium tuberculosis (including type strains of the virulent H37Rv and avirulent H37Ra organisms) as well as from M. bovis (one strain), M. bovis BCG (two strains), M. africanum (eight strains) and M. xenopi (one strain) but not from M. microti (one strain). The mycobactins from the tuberculosis group (M. tuberculosis, M. bovis and M. africanum) were identical and could each be resolved into four compounds by thin-layer chromatography (TLC) and into several fractions by high-performance liquid chromatography, supporting previous claims that this group is a single taxon. Exochelins were all chloroform-soluble and showed no species specificity; no single exochelin was recognized by TLC that had not been previously seen in M. avium or a related species.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.     

In vivo modulation of atypical mycobacterial infection: adjuvant therapy increases resistance to Mycobacterium avium by enhancing macrophage effector functions

Susceptible BALB/c mice were infected iv with a strain of Mycobacterium avium and infused with different biological response modifiers (BRM) in a gel delivery system so as to modify the progression of the infection in a beneficial fashion. Infusion of IL-2 or IL-4 in hydrophobic gels led to no significant enhancement of resistance. Infusion of muramyl dipeptide in hypromellose led to a significant enhancement of resistance against the M. avium, as seen by a significant reduction of colony-forming units (CFU) in the spleens of infected mice. Similarly, infusion of interleukin-1 beta in hypromellose in infected mice led to a significant reduction in CFU counts in the organs of mice. The mechanism(s) responsible for this enhanced resistance was studied further. It was found that infected mice developed profound immunosuppression, as judged by mitogenic and antigenic stimulation. Mice infused with MDP/hypromellose developed a similar immuno-suppression, suggesting that this adjuvant immunotherapy did not act by stimulating a T-cell response or by abrogating a putative suppressive phenomenon. Macrophages from mice infused with MDP alone were no more bacteriostatic for a virulent M. avium than control cells. However, macrophages from infected mice infused with MDP/hypromellose were more bacteriostatic for M. avium than cells from mice infected with M. avium and infused with the hydrophobic gel only. Overall, these results suggest that adjuvant immunotherapy is beneficial in M. avium infections.     

Inhibitory effect of IL-1 on the TSH dependent growth of rat thyroid cells (FRTL-5)

Using a functioning rat thyroid cell line (FRTL-5), we examined the effects of some cytokines, particularly interleukin-1 (IL-1) on the growth of thyroid cells. In 5H medium, namely Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a five-hormone preparation consisting of insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lysine acetate and somatostatin, IL-1 enhanced the growth of FRTL-5 cells detected by [3H]TdR incorporation. However, in 6H medium (5H medium plus bovine TSH), IL-1 inhibited the growth of FRTL-5 cells. Both effects were neutralized by the addition of anti-IL-1 antibody. Furthermore, IL-1 inhibited the growth of FRTL-5 cells induced by forskolin which is known as an adenylate cyclase activator. FRTL-5 cells have specific IL-1 receptors detected by the binding of 125I-labeled IL-1 alpha. By Scatchard plot analysis, the numbers and the dissociation constants of IL-1 receptors on FRTL-5 cells were shown to be 5225/cell and 8.69 x 10(-10) M. Interleukin-2, interleukin-6 and interferon-gamma (IFN-gamma) had no significant effects on the cell growth in 6H medium, while IFN-gamma and insulin-like growth factor I stimulated cell growth somewhat in 5H medium. These results suggest that IL-1 plays a regulatory role in the growth of thyroid cells through binding to the IL-1 receptors.     

Induction of murine macrophage TNF-alpha synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid

We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-alpha expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-alpha levels. Blockage of the CR4 iC3b site yielded increases in TNF-alpha for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-alpha only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-alpha expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways.     

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.     

Factors influencing the chlorine susceptibility of Mycobacterium avium,Mycobacterium intracellulare,and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Transforming growth factor beta1 blocks the release of collagen fragments from boving nasal cartilage stimulated by oncostatin M in combination with IL-1alpha

Oncostatin M in combination with interleukin-1 (IL-1) induced a rapid and reproducible release of collagen from bovine nasal cartilage in culture. This release was accompanied by a high collagenolytic activity and low or absent tissue inhibitor of metalloproteinase-1 activity in the culture medium. Transforming growth factor-beta1 was able to block this release of collagen from the tissue, and reduce the expression and secretion of collagenases whilst maintaining TIMP-1 levels from bovine nasal chondrocytes. This study shows for the first time that TGF-beta1 can protect cartilage collagen from destruction.     

Purification and characterization of soluble human IL-6 receptor expressed in CHO cells

An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.     

Protein kinase C-linked inactivation of the interleukin-1 receptor in a human transformed B-cell line

The effect of tumor-promoting phorbol ester treatment on the binding of interleukin-1 beta (IL-1 beta) to specific cell surface receptors was investigated. A 1 h exposure of Raji human B lymphoma cells with the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu), reduced IL-1 beta binding by up to 90% of control cells. This effect was dose-dependent and was not observed with 4-alpha-phorbol, an inactive tumor promoter. Analysis of 125I-labeled IL-1 beta binding to intact cells revealed that PDBu caused a 91% decrease in high-affinity cell-surface receptor number without an effect on receptor affinity. The phorbol ester response was rapid (30 min), observed both at 4 and 37 degrees C, and was preceded by the rapid translocation (t much less than 6 min) of protein kinase C (PKC) from the cytosol to the cell membrane. The PDBu-induced decrease in IL-1 beta receptor number was inhibited by prior incubation of cells for 30 min with the PKC inhibitor 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine (H7). The decrease in receptor binding was not due to enhanced IL-1 beta receptor internalization or shedding into the extracellular medium, since a similar effect was observed with solubilized IL-1 beta receptor. The most likely explanation for the phorbol ester effect appears to be cell surface inactivation of IL-1 receptors. These data suggest that modulation of PKC activity could play a role in the regulation of the IL-1 beta receptor.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.